effect of baﬁlomycin a1 and nocodazole on endocytic...

JOURNAL OF VIROLOGY,0022-538X/98/$04.0010

Dec. 1998, p. 9645–9655 Vol. 72, No. 12

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Effect of Bafilomycin A1 and Nocodazole on EndocyticTransport in HeLa Cells: Implications for Viral

Uncoating and InfectionNORA BAYER,1 DANIELA SCHOBER,1 ELISABETH PRCHLA,1 ROBERT F. MURPHY,2

DIETER BLAAS,3 AND RENATE FUCHS1*

Department of General and Experimental Pathology1 and Institute for Biochemistry,3 University of Vienna,Vienna, Austria, and Department of Biological Sciences, Carnegie Mellon University,

Pittsburgh, Pennsylvania 152132

Received 1 June 1998/Accepted 9 September 1998

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstratethe requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects thetransport of endocytosed material from early to late endocytic compartments. To characterize the endocyticroute in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled humanrhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up viareceptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of

46). This structural modification of the viral capsid is a pre-requisite for translocation of the viral RNA across the endo-somal membrane and thus for successful infection. Studieswith baf have revealed that the conformational change and theinfection was solely dependent on the activity of the endosomalv-ATPase (52). The pH threshold of the structural modifica-tion of the viral capsid in vitro is 5.6 (20). Thus, in vivo, viraluncoating is restricted to late endocytic compartments (52).This idea is supported by the accumulation of RNA-free 80Ssubviral particles in isolated late endosomes by 10 min afterinfection. Further transport of HRV2 from late endosomes tolysosomes was monitored by lysosomal degradation of its cap-sid proteins that occurs 30 min after uptake (32, 45, 52).

In the past few years baf has proved to be an excellent toolfor demonstrating the dependence of viral infectivity on lowendosomal pH (see, for example, references 36 and 51). How-ever, since baf might also affect transport through the endoso-mal system, inhibition of infection could likewise result fromtrapping of the virus in an endosomal subcompartment whichlacks components essential for uncoating. Nocodazole doesnot affect endosomal pH but has been reported to inhibitendosomal transport from ECV to late endosomes. The drugmight trap virus in ECV and thus allow determination ofwhether uncoating is only possible from later endocytic com-partments. The endocytic traffic of HRV2 and fluid-phasemarkers in HeLa cells in the presence or absence of baf andnocodazole was therefore investigated. By using fluorescencemicroscopy and subcellular fractionation by free-flow electro-phoresis, we show that the transport of all tracers from early tolate endosomes was blocked by inhibition of endosomal acid-ification. Transfer from early to late endosomes depended onmicrotubules and required ECV. The pH in these ECV wasfound to be similar to the pH in late endosomes (pH , 5.6).Since HRV2 infection proceeded normally in the presence ofnocodazole, successful uncoating can thus take place in ECV.Taken together, baf and nocodazole can thus be applied toarrest endocytic transport processes in different subcompart-ments in HeLa cells.

MATERIALS AND METHODS

Chemicals. All chemicals were obtained from Sigma unless specified. baf,kindly provided by K. H. Altendorf, University of Osnabrück, Osnabrück, Ger-many, was dissolved in dimethyl sulfoxide (DMSO) at 20 mM and stored at220°C. Nocodazole was dissolved in DMSO at 6 mg/ml. The final concentrationof DMSO, which was also added to control samples, was kept below 1%. Fluo-rescein isothiocyanate (FITC)-conjugated transferrin was prepared as describedpreviously (56). FITC-dextran (FD 70) was extensively dialyzed against Tris-buffered saline pH 7.4 and finally against phosphate-buffered saline (PBS) beforeuse. Lysine-fixable FITC-dextran (Mr 5 10 kDa), tetramethylrhodamine isothio-cyanate (TMR) conjugated dextran and TMR-conjugated bovine serum albumin(TMR-BSA) were purchased from Molecular Probes (Eugene, Oreg.). FITC andTMR derivatives dissolved in PBS were diluted 1:5 in Leibowitz 15 medium (L15;Life Technologies, Vienna, Austria). Moviol 4-88 was purchased from Calbio-chem and used at a concentration of 10% in aqua dest. Cy5.18-OSu (Cy5) wasobtained from Amersham and coupled to dextran (Mr 5 70 kDa) as describedearlier (59). [35S]methionine was obtained from ARC, St. Louis, Mo. LyophilizedStaphylococcus aureus cells (IgG-Sorb) were from The Enzyme Center, Malden,N.M. The polyclonal antibody against the cation-independent mannose-6-phos-phate receptor (Man6P-R) was a kind gift of B. Hoflack (Institute Pasteur, Lilles,France).

Cell culture and virus propagation. HeLa cells (Wisconsin strain, kindly pro-vided by R. Rueckert, University of Wisconsin) were grown in monolayers inminimal essential medium (MEM)-Eagle (GIBCO) containing heat-inactivated10% fetal calf serum; in suspension culture Joklik’s MEM (GIBCO) supple-mented with 7% horse serum was used. HRV2 was propagated, labeled with[35S]methionine, and purified as described earlier (65). 3H-labeled poliovirustype 2 Sabin, prepared as described previously (15), was a kind gift of PeterKronenberger.

Fluorescence microscopy. Cells were plated at low density on plastic 8-wellchamber slides the day before the experiment. To investigate the effect of baf onendocytic transport of FITC-dextran (Mr 5 10 kDa), cells were preincubated inserum-free L15 medium for 30 min at 37°C in the absence or presence of 20 nM

or 200 nM baf; then FITC-dextran was added at 10 mg/ml, and incubation wascontinued for 25 min. To determine the influence of nocodazole on endocytictransport, cells were preincubated as described above, and FITC-dextran wasadded at 10 mg/ml for 10 min at 37°C, followed by a 15-min chase. Finally, earlyendosomes were labeled for 5 min with 10 mg of TMR-dextran per ml at 37°C.Where indicated, 20 mM nocodazole was present throughout preincubation andendosome labeling. Alternatively, FITC-dextran (10 mg/ml) was internalized for10 min and chased for an additional 15 min in marker-free medium to label lateendosomes. Then, TMR-dextran was added to the medium for 5 min. Thereafter,cells were cooled to 4°C and incubated without or with 20 mM nocodazole for 60min. Finally, cells were warmed to 37°C for 5 min (with or without nocodazole).Labeled cells were cooled to 4°C, washed extensively with ice-cold PBS, fixedwith 4% paraformaldehyde for 1 h at room temperature, and quenched with 50mM NH4Cl in PBS. Cells were mounted in Moviol and viewed with an OlympusAH2 microscope. Kodak T-Max films (3200 ASA) were used for photography.

Immunofluorescence localization of Man6P-R in cells with internalized TMR-BSA. HeLa cells were preincubated without or with 20 mM nocodazole for 30min at 37°C. TMR-BSA (10 mg/ml) was added to the medium for 15 min, andcells were then incubated in marker-free medium (with or without nocodazole)for 10 min to label late endosomes and ECV, respectively. Subsequently, the cellswere cooled, fixed with 4% paraformaldehyde for 1 h at room temperature,quenched with 50 mM NH4Cl in PBS, washed three times with PBS (containing1% BSA), and incubated with a rabbit antiserum against Man6P-R (1:200 in PBScontaining 1% BSA and 0.005% saponin) for 20 min at room temperature. Thebound antibody was detected with FITC-conjugated mouse anti-rabbit immuno-globulin G (1:50 in PBS containing 1% BSA and 0.005% saponin), and cells weremounted in Moviol as described above.

Endosome labeling for subcellular fractionation. Cells (5 3 107/ml) werepreincubated for 30 min at 37°C with infection medium (MEM-Eagle containing2% fetal calf serum [FCS] and 30 mM MgCl2) in the absence or presence of therespective inhibitor (200 nM baf, 70 mM NH4Cl, or 20 mM nocodazole). 35S-labeled HRV2 (106 cpm) was added and internalized for 30 min at 34°C. Lateendosomes were labeled by incubation of 5 3 107 cells with FITC-dextran (20mg/ml) for 3 min at 37°C, followed by a chase of 12 min in marker-free medium.Horseradish peroxidase (HRP) was then added to a final concentration of 10mg/ml, and cells were incubated for 3 min to label early endosomes. Alterna-tively, FITC-transferrin was used as an early or a recycling endosome marker,while early endosomes and late endosomes were labeled by continuous internal-ization of HRP. HeLa cells were washed with PBS and incubated with serum-freeMEM-Eagle medium for 30 min to deplete endogenous transferrin. Cells werethen allowed to internalize 20 mg of FITC-transferrin per ml for 30 min at 37°Cand HRP for 25 min. Labeled cells were rapidly cooled and washed with coldPBS containing 10 mM EDTA to remove surface-bound HRV2 (31). Addition-ally, poliovirus that forms a stable complex with its receptor (29) was used tolabel plasma membranes. 3H-labeled poliovirus (3 3 106 cpm) was bound to thecells in PBS for 60 min at 4°C, and unattached virus was removed by repeatedwashing with PBS.

Preparation of endosomes by free-flow electrophoresis. As indicated in therespective figure legends, cells labeled with virus and endocytic marker weremixed and homogenized in 4 volumes of 0.25 M sucrose in TEA buffer (10 mMtriethanolamine, 10 mM acetic acid, and 1 mM EDTA, titrated with NaOH topH 7.4) with a ball bearing homogenizer (4). Nuclei and unbroken cells werepelleted (1,000 3 g, 10 min) to obtain the postnuclear supernatant. Microsomeswere prepared by spinning the postnuclear supernatant onto a 2.5 M sucrosecushion in a TST 60.4 rotor at 100,000 3 g for 1 h. The pellet was resuspendedand adjusted to 1 mg of protein per ml in 0.25 M sucrose in TEA buffer. Thesample was then subjected to gentle trypsin treatment by incubation with 3%TPCK trypsin/mg protein for 5 min at 37°C (37). The reaction was stopped byadding a tenfold excess of soybean trypsin inhibitor at 4°C. Microsomes wereinjected (1 ml/h) into a Bender and Hobein Elphor Vap 22 free-flow electro-phoresis apparatus at 120 mA and 1,300 V with 0.25 M sucrose in TEA buffer inthe chamber (37, 60, 61). A total of 92 fractions were collected (3 ml/fraction andat a rate of 3 ml/h) and assayed for protein content (6), radioactivity, fluores-cence, and HRP activity.

Endosome labeling for flow cytometry. To investigate the effect of baf onendosomal pH, two different protocols were applied. In the first, HeLa suspen-sion cells (108) were preincubated in 2 ml of Dulbecco MEM (DMEM) with orwithout baf for 30 min at 37°C. Cells were then incubated in fresh mediumcontaining 6 mg of FITC-dextran and 1 mg of Cy5-dextran per ml with or withoutbaf for 5 min at 37°C and chased in fresh medium with or without baf for 15 or30 min. In the second protocol, HeLa cells were continuously labeled for 25 minwith FITC-dextran and Cy5-dextran as in protocol 1; baf was then added to onealiquot, and incubation was continued in marker-free medium. For nocodazoletreatment, HeLa cells (108) grown in suspension were pelleted and preincubatedin 4 ml of suspension medium for 30 min at 37°C with or without 20 mMnocodazole. Cells were resuspended in 2 ml of DMEM containing 10% FCS withor without nocodazole. Endosomes were labeled by the addition of 6 mg ofFITC-dextran and 1 mg of Cy5-dextran per ml for 5 or 15 min at 37°C, followedby incubation in marker-free medium (2 ml of DMEM containing 10% FCS) forthe times indicated in the figure legends. Internalization was halted by theaddition of ice-cold PBS (pH 7.4), pelleting of the cells, and washing the pellet

9646 BAYER ET AL. J. VIROL.

twice with PBS. The pellet was resuspended in PBS and analyzed immediately byflow cytometry.

Generation of pH standard curves of internalized markers by flow cytometry.Cells labeled for 5 or 15 min (see above) were pelleted and divided into eightaliquots. These were resuspended in standard pH buffers. Buffers of the desiredpH (between 5.0 and 7.5) were obtained by mixing 50 mM HEPES with 50 mMmorpholineethanesulfonic acid MES (both containing 50 mM NaCl, 30 mMammonium acetate, and 40 mM sodium azide), accordingly. The samples wereleft on ice for 5 min for ATP depletion and for equilibration of intravesicular pH(pH clamped).

Flow cytometry. A dual-laser FACS-Calibur (Becton Dickinson Immunocy-tometry Systems) equipped with argon ion and red-diode lasers was used. FITCfluorescence (488-nm excitation) was determined by using a 530-nm band passfilter (30-nm band width), and Cy5 fluorescence (635-nm excitation) was deter-mined by using a 661-nm band pass filter (16 nm band width).

Calculation of intravesicular pH. Each sample was analyzed eight times, andpH clamped samples were determined as duplicates. The mean fluorescencevalues of FITC-dextran and Cy5-dextran were calculated for each sample, andthe autofluorescence from unlabeled samples was subtracted. The ratio of FITCto Cy5 was determined, and the intravesicular pH was calculated from thestandard curve.

Immunoprecipitation of HRV2 from infected cells. Cells (5 3 105) were incu-bated in 500 ml of infection medium (MEM supplemented with 2% FCS and 30mM MgCl2) at 4°C with or without 20 mM nocodazole for 30 min. Cells wereresuspended in fresh infection medium with or without inhibitor, incubatedunder slow rotation in a water bath at 34°C with 105 cpm of 35S-labeled HRV2for the times indicated, pelleted, and washed three times with PBS–10 mMEDTA at 4°C to remove cell-surface-bound virus. Cell pellets were lysed in 300ml of radioimmunoprecipitation assay (RIPA) buffer (9) for 10 min at 4°C, andthe cell debris was removed by centrifugation for 10 min in an Eppendorfcentrifuge. Cell pellets and supernatants were processed separately for S. aureus-aided immunoprecipitation with monoclonal antibody 2G2 (specific for C anti-gen), followed by rabbit anti-HRV2 hyperimmune serum (specific for both C andD antigens) as described earlier (45). Radioactivity in the immunoprecipitateswas determined by liquid scintillation counting. Alternatively, pellets were boiledin Laemmli sample buffer and analyzed on 10% polyacrylamide-sodium dodecylsulfate (SDS) mini-gels, followed by fluorography.

Viral protein synthesis. Suspension cells (5 3 105) in 500 ml of methionine-free infection medium were preincubated for 30 min with or without baf ornocodazole and challenged with HRV2 at a multiplicity of infection (MOI) of500 at 34°C. At 4 h postinfection, the medium was replaced with fresh methi-onine-free infection medium (with or without inhibitor) containing 20 mCi of[35S]methionine, and incubation was continued overnight. Cells were pelletedand lysed in 300 ml of RIPA buffer, and viral proteins were immunoprecipitatedwith rabbit antiserum against HRV2, boiled in Laemmli sample buffer, andseparated on 10% polyacrylamide-SDS minigels. Viral proteins VP1 throughVP3 of the progeny virus were detected by autoradiography. Data from a single,representative experiment are shown. All experiments were repeated three timeswith similar results.

RESULTS

Transport from early to late endosomes in HeLa cells isdependent on low endosomal pH. First, it was verified whetherbaf indeed elevates the pH in endocytic compartments ofHeLa cells to neutrality. Cells were incubated for 30 min at37°C in the absence or presence of 200 nM baf, and the accu-mulation of acridine orange (1 mM for 10 min at 37°C) wasinvestigated by fluorescence microscopy. In control cells, thedrug accumulated in endosomes and lysosomes, which wasindicative of acidic luminal pH, whereas no vesicle staining wasseen in the presence of the drug; this finding demonstratesinhibition of vesicle acidification (data not shown). Alterna-tively, endosomes were labeled for 5 min by internalizationwith a pH-sensitive (FITC) and pH-insensitive (Cy5) derivativeof a fluid-phase marker (dextran), and the pH of labeled com-partments was calculated (for various chase times) from theratio of the fluorescence intensities as analyzed by flow cytom-etry. After a 30-min chase, the average pH of labeled compart-ments was 6.3 in the absence and 7.4 in the presence of 200 nMbaf (Fig. 1A). Thus, at this concentration, baf efficiently blocksendosome acidification. To determine the time required todissipate endosomal pH gradients upon baf addition, endo-somes were continuously labeled for 25 min with FITC andCy5-dextran, whereupon baf was added for up to 60 min. Asshown in Fig. 1B, the addition of 200 nM baf for 30 min is

sufficient to raise the average pH of all labeled compartmentsto neutrality. In contrast, when baf was present at a 20 nMconcentration, the pH in endocytic compartments was onlyincreased by about 0.2 pH units under either labeling condition(Fig. 1).

To investigate the influence of baf on endocytic transport inHeLa cells by fluorescence microscopy, the fluid-phase markerFITC-dextran was internalized for 25 min at 37°C in the ab-sence or presence of 20 nM or 200 nM baf (see Materials andMethods). In control cells, vesicular accumulation of FITC-dextran resulted in a bright fluorescence staining in the perinu-clear region, which is characteristic of late endosomes. WhenFITC-dextran was internalized in the presence of 20 nM baf,no influence on the accumulation of marker in late endosomeswas observed. However, in the presence of 200 nM baf, verysmall peripheral vesicles were stained, resulting in a patterntypical for early endosomes (Fig. 2). To further identify thecompartments accessible to endocytic markers in the presence

FIG. 1. baf raises the pH of early and late endosomes in HeLa cells. (A)HeLa cells were preincubated for 30 min at 37°C without or with 20 nM or 200nM baf. They were then incubated for 5 min in medium containing 6 mg ofFITC-dextran and 1 mg of Cy5-dextran per ml and subsequently chased indextran-free medium in the absence or presence of baf. Cells were immediatelycooled, washed with PBS, and analyzed by flow cytometry. (B) HeLa cells werecontinuously labeled with 6 mg of FITC-dextran and 1 mg Cy5-dextran per ml for25 min. Cells were then further incubated in fresh medium without or with 20 nMor 200 nM baf (arrow). The average pH of labeled endocytic compartments wascalculated with a pH calibration curve (see Materials and Methods). Timesshown in both panels represent total time from the addition of FITC-dextran andCy5-dextran. Values shown are means and standard deviations from two exper-iments (each sample was analyzed eight times).

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of baf, free-flow electrophoresis was employed to separateendosome subpopulations (60, 62). Early and late endosomeswere labeled by pulse-chase with the fluid-phase markers HRPand FITC-dextran, respectively (see Materials and Methods).Furthermore, HRV2 was used as a pH-sensitive, receptor-dependent ligand (52, 53). Virus was taken up into HeLa cellsfor 20 min at 34°C, conditions expected to lead to accumula-tion in early and late endosomes. In order to demonstrate thedistribution of bona fide plasma membranes (plasma mem-brane enzymes are also found in endosomes [37, 60]), 3H-labeled poliovirus was bound to an aliquot of the cells at 4°C tolabel plasma membranes. The association of poliovirus with itsplasma membrane receptor is stable under the conditions offree-flow electrophoresis (28). To avoid interference of fluid-phase markers and viruses, labeling of HeLa cells with thefluid-phase markers was carried out independently and cellspreincubated with the various compounds were mixed beforehomogenization. Microsomes were prepared and subjected toFFE after gentle trypsin treatment, which is required toachieve endosome separation (37, 61). Plasma membranes la-beled with 3H-poliovirus were well separated from anodallyshifted endosome fractions (Fig. 3A). HRP activity was mainlyrecovered in a slightly shifted peak representing early endo-somes. The more anodally shifted peak of HRP activity reflectsthe arrival of the marker in late endosomal compartmentsalready after 3 min of internalization. This finding is in agree-ment with a rapid transit through the early compartments (60).Although continuously internalized, the majority of HRV2 ac-cumulated in late endosomes together with FITC-dextran. Thismight be due to the smaller volume of early endosomes com-pared to late endosomes. However, when HRV2 and fluid-phase markers had been taken up in the presence of baf,HRV2, HRP, and FITC-dextran accumulated in a compart-ment exhibiting the same electrophoretic mobility as the earlyendosomes (Fig. 3B). A similar distribution of internalizedmarkers was observed when the vesicular pH was elevated bythe presence of 70 mM NH4Cl instead of baf (data not shown).Taken together, these results clearly demonstrate that com-plete inhibition of endosome acidification leads to the arrest ofendocytosed ligand and fluid-phase marker in early endo-somes. In the case of HRV2 this might be also due to inhibitionof low-pH-dependent receptor-ligand dissociation.

Finally, the influence of different baf concentrations onHRV2 infection was determined. Cells were preincubatedwithout or with 20 nM or 200 nM baf and then infected withHRV2. Newly synthesized viral proteins were detected by la-beling of progeny virus with [35S]methionine. In agreementwith our previous data (62), 200 nM baf completely inhibitedthe infection of HeLa cells by HRV2 (Fig. 4). Although 20 nMbaf failed to raise endosomal pH to neutrality and to blockendocytic transport (Fig. 1 and 2), viral infection was pre-vented (Fig. 4). This can be explained by the pH thresholdrequired for the conformational change of HRV2 capsid pro-teins that is a prerequisite for infection (45, 52). As demon-strated by Gruenberger et al. (20) maximum conversion ofnative virus to conformationally altered C-antigenic particlesoccurs below pH 5.6. Increasing the pH by 0.2 U results in a60% inhibition of this conformational change in vitro. Thus,elevation of endosomal pH by about 0.2 U in vivo appears tobe sufficient to prevent HRV2 uncoating and infection (Fig. 4).

Influence of nocodazole on endocytic transport in HeLacells. The transfer of endocytosed material from early to lateendosomes has been shown to be blocked by microtubule-depolymerizing agents (5, 18). We therefore investigated theinfluence of nocodazole on endocytic transport in HeLa cells.First, we confirmed the breakdown of the microtubule network

FIG. 2. Influence of baf on endocytic transport of FITC-dextran as analyzedby fluorescence microscopy. HeLa cells grown on chamber slides were preincu-bated without or with baf (20 nM or 200 nM) in L15 medium for 30 min at 37°C.Ten milligrams of FITC-dextran per ml was added, and endosomes were labeledfor 25 min at 37°C. Cells were cooled, rinsed with PBS, fixed with 4% parafor-maldehyde, quenched with 50 mM NH4Cl in PBS, and viewed with an Olympusfluorescence microscope.


after treatment with 20 mM nocodazole for 30 min at 37°C byindirect immunofluorescence microscopy with an FITC-la-beled anti-a-tubulin antibody (data not shown). Next, the in-tracellular traffic of endocytic markers in the presence ofnocodazole was investigated by fluorescence microscopy.FITC-dextran was chased into late endosomes, whereas TMR-dextran was internalized into early compartments. In untreatedcells most of the TMR-dextran is localized in small punctateand tubular vesicles well separated from the larger, perinuclearlate endosomes labeled with FITC-dextran (Fig. 5A, upperpanels). Nocodazole-treated cells displayed a peripheral distri-bution and some colocalization of FITC-dextran and TMR-dextran (see arrowheads in Fig. 5A, lower panels). However,many compartments were accessible to FITC-dextran but notto TMR-dextran (see arrows in Fig. 5B, lower panels); theseare presumed to represent ECV, which have been shown toaccumulate under these conditions (18). In agreement with theresults of Gruenberg and Howell (18) for BHK cells, the trans-

port of endocytosed substances from early to late endosomesdepends on the presence of intact microtubules in HeLa cellsas well.

Since the distribution of late endosomes and lysosomes isalso affected when microtubules are disrupted (21, 38), weverified that fluid-phase markers were indeed accumulated inECV in the presence of nocodazole. First, late endosomeswere labeled by pulse-chase with FITC-dextran, and TMR-dextran was subsequently internalized for 5 min to label theearly endosomes. Cells were then cooled to 4°C, incubated for1 h in the absence or presence of nocodazole, and subsequentlywarmed to 37°C for 5 min. This short incubation period wassufficient to transfer TMR-dextran from early endosomes tolate endosomes in the absence of nocodazole (60, 62), resultingin colocalization of both markers in perinuclear late compart-ments (Fig. 5B, arrowheads in upper panels). Disruption ofmicrotubules led to the accumulation of TMR-dextran in largevesicles (ECV) in the cell periphery. Although late endosomeslabeled with FITC-dextran had acquired a random distributionin the vicinity of ECV under this condition, they are clearlydistinct from ECV (Fig. 5B, lower panels). Second, the influ-ence of nocodazole on colocalization of internalized markerwith the cation-independent Man6P-R, which is primarilyfound in late endosomes and in the trans-Golgi network (21,38), was investigated. For this purpose, TMR-BSA was inter-nalized into late compartments of control or nocodazole-treated cells. Cells were then fixed and permeabilized, andlocalization of Man6P-R was revealed by indirect immunoflu-orescence. Under control conditions, considerable colocaliza-tion of endocytosed marker and Man6P-R in perinuclear com-partments was seen (Fig. 5C, upper panels). However, uponinternalization of the marker in the presence of nocodazole,transfer to Man6P-R-positive structures was prevented (Fig.5C, lower panels). Again, nocodazole treatment resulted in aredistribution of the Man6P-R-labeled structures to the cellperiphery. Nevertheless, the compartment where the marker isaccumulated when microtubules are disrupted is clearly dis-tinct from the late endosomes.

Luminal pH of ECV. So far, the luminal pH of those vesicleswhich accumulate internalized material in the presence of no-codazole (ECV) has not been determined. Therefore, kineticanalysis of endosome acidification in living cells was carriedout by flow cytometry, exploiting the pH dependence of FITC-dextran fluorescence. To determine the pH of selectively la-beled compartments, two protocols were applied. In the first,early endosomal compartments were labeled by a short pulse

FIG. 3. baf inhibits delivery of HRV2 and fluid-phase markers to late endo-cytic compartments. HeLa cells preincubated without (A) or with (B) 200 nM baf(30 min, 37°C) were divided into three aliquots each. (i) Late endosomes werelabeled with FITC-dextran (20 mg/ml; 3-min pulse, 12-min chase in marker-freemedium); HRP (10 mg/ml) was then added for 3 min at 37°C to label the earlyendosomes. (ii) 35S-labeled HRV2 (106 cpm) was internalized for 20 min at 34°C.(iii) 3H-labeled poliovirus (6.5 3 105 cpm) was bound to HeLa cells for 60 minat 4°C to label plasma membranes. The aliquots of the labeled cells were cooledand combined, washed with PBS containing 10 mM EDTA to remove plasmamembrane bound HRV2, and homogenized in TEA-sucrose buffer (see Mate-rials and Methods). Microsomes were analyzed by FFE. A total of 92 fractionswere collected, and protein content, radioactivity, and HRP activity, were deter-mined. Data are expressed as the percentage of the total amount recovered afterFFE.

FIG. 4. baf prevents infection of HRV2. HeLa cells were preincubated ininfection medium (with or without 20 or 200 nM baf) for 30 min at 34°C. HRV2was added at an MOI of 500. At 4 h postinfection, the medium was replaced withfresh methionine-free medium containing 200 mCi of [35S]methionine, and in-cubation was continued overnight. Cells were collected by centrifugation andlysed, and newly synthesized viral proteins were immunoprecipitated withanti-HRV2 antiserum. Viral proteins were analyzed by polyacrylamide gel elec-trophoresis followed by autoradiography. Where indicated, baf was presentthroughout the experiment.


FIG. 5. Effect of nocodazole on fluid-phase marker transport in HeLa cells. (A) Cells on chamber slides were incubated without or with 20 mM nocodazole inserum-free MEM (30 min, 37°C). Late endosomes were labeled by pulse (10 min)-chase (15 min) with 10 mg of FITC-dextran per ml followed by early endosomelabeling with 10 mg of TMR-dextran per ml for 5 min, and the cells were prepared for fluorescence microscopy. Where indicated, nocodazole was present throughout.FITC and TMR fluorescence images of the same cells are shown. Arrows indicate ECV; arrowheads indicate colocalization of markers in early endosomes. (B) Lateendosomes were labeled by pulse (10 min)-chase (10 min) with 10 mg of FITC-dextran per ml followed by TMR-dextran (10 mg/ml) internalization for 5 min. Cellswere rapidly cooled and incubated in the absence (upper panels) or presence of nocodazole for 1 h at 4°C before being warmed to 37°C for 5 min. In the absence ofnocodazole, both markers colocalized in late endosomes (arrowheads, upper panels). When cells were treated with nocodazole after labeling of early and lateendosomes, transfer of TMR-dextran to late, FITC-labeled endosomes (arrows, lower right panel) was arrested in EVC (arrows, lower left panel). (C) Cells werepreincubated without or with nocodazole as in panel A, followed by internalization of 10 mg of TMR-BSA per ml for 15 min and a 10-min chase in marker-free medium(with or without nocodazole). Cells were cooled and fixed, and Man6P-R was detected by indirect immunofluorescence. In control cells, TMR-BSA was mainly detectedin Man6P-R-positive compartments (late endosomes [arrowheads, upper panels]). Disruption of microtubules arrested the transport of TMR-BSA in ECV (arrows,lower right panel), resulting in a considerable reduction of colocalization with Man6P-R-containing structures (late endosomes [arrows, lower left panel]).

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(2 min) with FITC-dextran followed by a chase in marker-freemedium for 15 min. In the second, late compartments (prely-sosomes and lysosomes) were labeled by a pulse for 15 minfollowed by a chase for 2 h. As shown in Fig. 6A, the pH ofFITC-dextran-labeled compartments decreased to 6.7 within 5min and to pH 6.0 within 20 min after internalization. Depo-lymerization of microtubules with nocodazole did not signifi-cantly affect the pH of the compartments reached by FITC-dextran between 5 and 20 min after internalization. Moreover,endosomes continuously labeled for 15 min (protocol 2; Fig.6B) had a similar pH of about 6.5 regardless of the presence ofthe drug. When the dextran was chased into prelysosomes andlysosomes, a pH of 5.2 was found for control cells. This is ingood agreement with published data (43, 55). However, in thepresence of nocodazole a pH of only 6.2 was attained after a120-min chase (Fig. 6B). This clearly demonstrates that no-codazole arrests transfer from compartments with an averagepH of 6.0 to compartments of pH 5.2 (lysosomes). It should be

noted that the values reported here may reflect an average formore than one type of compartment, since fluid-phase markersare expected to nonspecifically label all endocytic structures,including pH-neutral (recycling) endosomes (40, 64, 70) underthe conditions applied.

FIG. 6. Effect of nocodazole on the pH of endocytic compartments. HeLacells were preincubated (with or without 20 mM nocodazole) in DMEM for 30min at 37°C. (A) Endosomes were labeled with FITC (6 mg/ml)- and Cy5 (1mg/ml)-dextran in DMEM for 5 min and then chased for 15 min in marker-freemedium. (B) Cells were labeled for 15 min with FITC-dextran and Cy5-dextran(concentrations as in panel A) and chased for 120 min. The fluorescence inten-sity of the internalized markers was determined by flow cytometry. The averagepH of endocytic compartments was calculated as described in Materials andMethods. The times shown in both panels represent the total time from theaddition of FITC-dextran and Cy5-dextran. The values shown represent themeans and standard deviations of two independent experiments (each analyzedeight times).

FIG. 7. HRV2 is converted to C-antigenic particles but lysosomal degrada-tion is prevented in nocodazole-treated cells. (A and B) HeLa cells were prein-cubated (with or without 20 mM nocodazole) for 30 min at 34°C in infectionmedium and infected with [35S]HRV2 (105 cpm) at 34°C. Aliquots were removedat 5, 10, 20, and 30 min. Samples were rapidly cooled, cells were pelleted, plasmamembrane bound virus was removed with PBS–10 mM EDTA, and cell pelletswere lysed in RIPA buffer. Lysates (A) and cell supernatants (B) were subjectedto immunoprecipitation with monoclonal antibody 2G2, which is specific forC-antigenic particles, and polyclonal antiserum. (C) Cells were preincubated andinfected as in panel A for 30 min. Unbound virus was removed with PBS, andincubation was continued with or without nocodazol for the times indicated.Virus was recovered from cell lysates by immunoprecipitation with 2G2. Viralproteins were analyzed by SDS-gel electrophoresis followed by autoradiography.


To determine whether ECV can lower their luminal pHbelow 5.6, we took advantage of the low-pH-dependent con-formational change of the HRV2 capsid that results in theformation of C-antigenic particles. Upon exposure of the virusto pH of ,5.6, an epitope becomes exposed which is recog-nized by the monoclonal antibody 2G2 (20, 45). Consequently,the kinetics of the conformational modification of internalized[35S]HRV2 was assessed in cell lysates and supernatants. C-antigenic virions were immunoprecipitated with 2G2 followedby polyclonal anti-HRV2 rabbit hyperimmune serum (to re-cover native virus). Radioactivity in the precipitates was thendetermined in a beta counter. Conversion to C antigenicity,which occurred by 5 min after uptake, was insignificantly in-fluenced by nocodazole treatment (Fig. 7A). However, theamount of recycling of C-antigenic material to the cell super-natant was reduced by the drug (Fig. 7B). These findings implythat the pH of ECV accumulating HRV2 in the presence ofnocodazole is at least 5.6. This is in agreement with results byKillisch et al. (25), who concluded from indirect data that ECVin erythroblasts are more acidic than early endosomes. For thefirst time, we present here direct evidence to confirm andextend these data to HeLa cells.

Further proof for inhibition of endosomal transport to lyso-somes was obtained by determining the breakdown of viralproteins. The appearance of cleavage products (in particular ofVP1 [20]) of HRV2 capsid proteins after 30 min of internal-ization at 34°C has been demonstrated to be due to lysosomaldegradation, since the capsid proteins remain intact upon in-fection at 20°C (45). Under this condition the transfer fromlate endosomes to lysosomes is blocked (27). When HRV2 wasinternalized for 30 min and then chased for up to 90 min at34°C, no degradation of viral proteins in the presence of no-codazole was evident (Fig. 7C); thus, transfer to late endocyticcompartments (lysosomes) is clearly blocked by the drug.

HRV2 infection occurs in the presence of nocodazole. Hav-ing demonstrated that HRV2 is efficiently converted to C-antigenic particles in the presence of nocodazole, we nextsought to determine whether this results in productive infec-tion. HRV2 was internalized into HeLa cells and de novo-synthesized viral proteins were labeled with [35S]methionine.As revealed in Fig. 8, viral replication occurred normally in thepresence of nocodazole. Given that the transport from early tolate endosomes is inhibited by the presence of the drug (Fig.5), our data thus demonstrate productive uncoating of HRV2from ECV.

Properties of ECV. As shown by fluorescence microscopy,fluid-phase markers accumulate in ECV in the presence ofnocodazole (see Fig. 5). We thus wondered whether ECV canbe separated from early and late endosomes by using FFE.

Early and recycling endosomes were labeled with FITC-trans-ferrin while [35S]HRV2 and HRP were continuously internal-ized for 20 min in the presence of nocodazole to accumulatemarker in ECV. Microsomes were prepared and separated byFFE. Early and late endosomes were well separated from eachother; however, the distribution of endocytic markers fromcontrol (compare with Fig. 3A) and from nocodazole-treatedcells (Fig. 9) was indistinguishable. Accordingly, ECV haveelectrophoretic properties similar or identical to those of lateendosomes and so cannot be separated by FFE. Consequently,this technique, as well as density gradient centrifugation (2),cannot be applied to ECV isolation.

DISCUSSION

Using fluorescence microscopy and subcellular fractionationtechniques, we demonstrated that the v-ATPase inhibitor bafat a 200 nM concentration arrests transport of HRV2 andfluid-phase markers in early endosomes in HeLa cells. In con-trast, depolymerization of microtubules with nocodazole re-sulted in an accumulation in ECV (which may represent eithertransport vesicles or early endosomes arrested during matura-tion to form late endosomes). Since native HRV2 was modi-fied to its C-antigenic form in the presence of the drug, ECVmust exhibit a pH of at least 5.6. Recycling of C-antigenicparticles was somewhat reduced but was not prevented. ECVthus acidify their lumen to a pH almost as low as that found inlate endosomes and are able to recycle their cargo to theplasma membrane (Fig. 10).

baf effects on endocytic trafficking. v-ATPases establish andmaintain a low luminal pH in endocytic and exocytic compart-ments. These ATPases are specifically inhibited by the fungalmetabolite baf. As a consequence of this inhibition, this drughas been reported to also exert secondary effects such as (i)inhibition of receptor-ligand dissociation (22), (ii) altered traf-ficking of transmembrane proteins (54), (iii) inhibition of bud-ding of ECV from early endosomes (8), (iv) inhibition of lateendosome-lysosome fusion (73), and (v) fragmentation of earlyendosomes (13). Most importantly, these secondary effects ap-pear to vary between different cell types. We thus asked

FIG. 8. HeLa cells are infected in the presence of nocodazole. Cells werepreincubated in infection medium with or without 20 mM nocodazole for 30 minat 34°C. HRV2 was added at an MOI of 500. At 4 h postinfection, the mediumwas replaced with fresh, methionine-free medium containing 200 mCi of [35S]me-thionine, and incubation was continued overnight. Cells were collected by cen-trifugation and then lysed; newly synthesized viral proteins were next immuno-precipitated with anti-HRV2 antiserum. Viral proteins were analyzed bypolyacrylamide gel electrophoresis followed by autoradiography. Where indi-cated, nocodazole was present throughout the experiment.

FIG. 9. ECV comigrate with late endosomes upon FFE separation. HeLacells were preincubated in serum-free MEM with or without (Fig. 3) 20 mMnocodazole for 30 min at 37°C. Aliquots of the cell suspension were labeled withFITC-transferrin (20 mg/ml, 30 min at 37°C), HRP (10 mg/ml, 3 min, 37°C), and35S-labeled HRV2 (106 cpm, 30 min at 34°C), respectively. Microsomes wereprepared and analyzed by FFE. Data are expressed as the percentage of the totalamount of the respective marker recovered after FFE.


whether baf also affected the endocytic route of fluid-phasemarkers in HeLa cells. First, the inhibition of vesicle acidifi-cation by baf was assessed. At a 200 nM concentration the drugcompletely inhibited vesicle acidification. However, at 20 nMthe mean vesicular pH was only increased by about 0.2 pH U,and the time-dependent decrease of endosomal pH was notprevented. We then demonstrated by fluorescence microscopythat this drug indeed leads to accumulation of fluid-phasemarkers in very small peripheral tubular endosomes that wereclearly not derived from late endosomes, since the addition ofbaf to FITC-dextran-loaded late endosomes did not alter theirdistribution. Such structures have been carefully characterizedin HeLa cells under normal conditions by electron microscopy(69). It should be noted that these effects of baf were only seenat 200 nM, a concentration which elevated the pH to neutral-ity. Accumulation of fluid-phase marker and HRV2 in earlyendosomes in baf-treated cells was also shown by separation ofendosome subpopulations by FFE. Although we cannot ex-clude with certainty that baf treatment alters the endosomalmembrane composition, leading to different electrophoreticproperties, the data are in accordance with the morphological

studies. As far as HRV2 is concerned we cannot differentiatebetween the effects of baf on receptor-virus dissociation andarrest of transport. Although C-antigenic (low pH modified)HRV2 particles do not bind to the HRV2 receptors (46), thepH threshold of dissociation of HRV2 from the LDL-a2-mac-roglobulin receptor family has not been determined. Never-theless, fluid-phase markers such as dextran and HRP werealso retained in early endosomes. Taken together, our resultsdemonstrate that the transport of endocytosed macromole-cules from early to late endosomes in HeLa cells depends onproper endosome acidification, regardless of whether the up-take occurs by fluid-phase or receptor-mediated endocytosis.This is in accordance with results in BHK, MDCK, and COScells, in macrophages, and in Dictyostelium discoideum (3, 8, 30,68). On the other hand, in cell lines in which maturation ofearly endosomes into late endosomes had been shown, bafprevented the delivery of material to lysosomes (72, 73). Re-cently, low-pH-dependent association of COP-I componentswith early endosomes was shown to be required for the for-mation of ECV (12, 21).

FIG. 10. Schematic representation of stages of endocytic traffic in HeLa cells blocked by baf and nocodazole. baf inhibits budding of ECV, leading to theaccumulation of cargo in early endosomes, whereas nocodazol depolymerizes microtubules, thus inhibiting the transport of ECV to late endosomes, but it has only aminor effect on recycling to the plasma membrane.


Consequences of the baf-induced transport block on viralinfection. We have previously shown that baf at 200 nM com-pletely blocks infection of HeLa cells by HRV2. Since we haveproven this acid dependence in vivo and in an in vitro systemin isolated endosomes (52, 53), it is legitimate to conclude thata low pH is a sine qua non condition for infection. Determi-nation of the mean vesicular pH in HeLa cells incubated with20 nM baf showed an increase of only 0.2 U (Fig. 1). Inagreement with a pH of ,5.6 required for structural modifi-cation of the viral capsid, this small pH increase was sufficientto inhibit viral infection. However, the results presented in thisreport call for cautious interpretation of a baf-induced block ofviral infectivity since cargo remains trapped in the early endo-somes in the presence of the drug, where factors required forpenetration might be lacking. Entry into a late endosomalcompartment could be an advantage for viruses requiring de-livery to the perinuclear region for uncoating (35). Most vi-ruses known to be uncoated along the endocytic pathway arepoorly characterized with respect to the specific subcompart-ment where the uncoating occurs. In addition, the variouseffects exerted by baf in a certain cell type are usually un-known. Thus, inhibition of virus infection by baf could eitherbe interpreted as low pH dependence and/or productive un-coating only taking place in late endocytic compartments.

Recycling to the plasma membrane in HeLa cells can occurfrom ECV. The subcellular localization of endocytic and exo-cytic compartments requires an intact and dynamic microtu-bular network. Consequently, microtubule depolymerizationdisrupts endocytic traffic at various stages, depending on thecell line under investigation (5, 71). Knowledge of the endo-cytic subcompartment which accumulates various markers thusallows the identification of the site of virus uncoating. Wepresent here evidence that in HeLa cells depolymerization ofmicrotubules by nocodazole blocks transport at the stage ofECV but only slightly inhibits recycling of modified HRV2subviral particles (see Fig. 7). This notion is in accordance withdata from Czekay et al. (11) demonstrating the recycling ofmegalin, a member of the LDL receptor family, from lateendocytic compartments.

Proposed model of endocytic transport and endosomal pHregulation in HeLa cells. The data presented here and else-where (38–40, 42) suggest the following concept for endosomalpH regulation. Macromolecules internalized via endocyticcoated vesicles that lack functional v-ATPases (16) are firstdelivered to mildly acidic early endosomes, where fast recy-cling of receptors (e.g., transferrin and LDL receptor) andplasma membrane proteins occurs via more-alkaline recyclingendosomes (64). Budding of ECV from early endosomes leadsto a pH decrease (5.6) in these compartments. Material inECV can then either be transferred to late endosomes andfurther to lysosomes or be recycled to the plasma membrane.Whether these recycling compartments maintain an acidic lu-men remains to be demonstrated. The maintenance of a mildlyacidic pH in early endosomes depends on electrogenic inter-action of the endosomal proton ATPase with Na1/K1-ATPase(7, 17, 58). It is thus conceivable that the budding of ECVexcludes the Na1/K1-ATPase, which remains in the tubularportions of the early endosome and is thus recycled back to theplasma membrane. Indeed, the absence of a functional Na1/K1-ATPase in late endosomes has been shown. It has to bepointed out, however, that Na1/K1-ATPase regulates endo-some acidification in some cell types (e.g., CHO [17], A549 [7],and Swiss 3T3 [74]) but not in others (e.g., K562 [63] and rathepatocytes [1]), again demonstrating differences in endocyticmembrane traffic that are dependent on cell specialization. Sofar, the roles of Na1/K1-ATPase and other factors in deter-

mining the steady-state pH of endocytic subcompartments inHeLa cells are unknown.

ACKNOWLEDGMENTS

We thank K. Altendorf and B. Hoflack for the kind gifts of baf andanti-Man6P-R antiserum, respectively.

This work was supported by Austrian Science Foundation grantsP10618-MED and P12967-GEN to R.F. and P12269-MOB to D.B.

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